Determination of urinary ortho- and meta-cresol in humans by headspace SPME gas chromatography/mass spectrometry

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Abstract

ortho-Cresol (o-C) and meta-cresol (m-C) are minor urinary metabolites of toluene, a widely used chemical with neurotoxicological properties. A new assay for their determination in human urine is here proposed. Urinary cresol sulphates and glucuronates are submitted to acid hydrolysis, urine is neutralized, added with o-cresols-d8, and analytes are sampled in the headspace of urine by SPME using a polydimethylsiloxane fiber. Analysis is performed by GC/MS using, for separation, either a SupelcoWax10 (for o-C) or a chiral CP Cresol (for o-C and m-C) column. The method is very specific, with a range of linearity 0–5.0 mg/l, within- and between-run precision, as coefficient of variation, <15% and <19%, limit of detection of 0.006 mg/l for o-C and 0.007 mg/l for m-C. The procedure is applied to the quantification of cresols in urine from workers exposed to toluene and from subjects belonging to the general population.

Introduction

Toluene is a very common chemical, which is widely used as a solvent in paints, coatings and many consumer products, as a synthetic intermediate and as an additive in fuel to increase gasoline octane rating [1].

Humans may experience toluene exposure in working settings and in living environments, being toluene a component of cigarette smoke, auto vehicular exhausted fumes, gasoline and various commodities vapours.

Due to its toxicity to the central nervous system, toluene exposure is regulated in many countries. An airborne concentration of 50 ppm (188 mg/m3) as occupational exposure limit during the work shift is recommended, suggested or established by industrial hygiene associations and/or governmental agencies in different countries [2], [3], [4], [5].

Following exposure in humans, toluene is readily transformed into several metabolites, among which the three isomers ortho-cresol (o-C), meta-cresol (m-C) and para-cresol (p-C). These compounds are excreted in urine as glucuronate and sulfate conjugates, accounting for few percent of the inhaled dose [6], [7].

For the biological monitoring of occupational exposure to toluene, o-C in urine is adopted as determinant both in the biological exposure indices (BEI) list by the American Conference of Governmental Industrial Hygienists (ACGIH) [2] and in the biological tolerance values for occupational exposures (BAT) list by the German Ministry for Employment and Social Affairs (AGS Committee for Hazardous Substances) [3]. A value of 0.5 mg/l for samples collected at the end of the shift is suggested as a BEI, and a BAT of 3.0 mg/l for samples collected at the end of the shift, after a long-term exposure, is listed [2], [3]. The “B” notation reported by ACGIH indicates that o-C is usually present in a significant amount in biological specimens collected from subjects who have not been occupationally exposed [2].

The use of urinary m-C for the same purpose, has never been investigated. On the contrary, the use of urinary p-C is precluded by the large amount of this chemical found in urine of all subjects, regardless the occupational exposure to toluene [7], [8].

Several analytical procedures for the determination of urinary o-C have been published so far, some of which, by means of a separation performed with either mobile or stationary chiral phase, are suitable also for m-C [7], [8], [9], [10], [11], [12], [13], [14]. Sample preparation is always required and usually performed, after hydrolysis of conjugates, by extraction of free cresols from urine using organic solvents [7], [8], [9], [10], [12], [15], [16], [17], [18], [19], [20], [21], vapour stream distillation [11], [22], [23] or solid phase using C18 reverse phase silica [24]. Direct headspace sampling of free cresols present in biological fluids following accidental or intentional ingestion is also proposed [14]. While the solvent extraction procedure, due to the high solubility of cresols in water, may be scarcely efficient, the vapour stream distillation needs large sample volume and may be poorly precise and/or affected by loss of analyte due to the use of relatively large distillation apparatus. Techniques such as HPLC and GC are applied for the separation of cresols from each other and/or from other chemicals, and UV, FID detectors are traditionally used for their quantification. The introduction of derivatization with fluorinated or fluorescent chemicals and consequent detection with ECD [7] or fluorimeter [12], as well as the use of MS detector [13], [23], have lowered the detection limit of the analytical techniques to few μg/l, allowing the determination of urinary cresols also in non occupationally exposed subjects.

In this work, an improved analytical assay suitable for the determination of a low level of cresols in human urine is presented. This assay is based on the volatility of free cresols that, after hydrolysis of their conjugates, are sampled in the headspace (HS) of urine by SPME and directly injected onto a proper capillary column for separation. The assay uses a small amount of specimen, performs all steps in one vial, introduces an internal standard, avoids the use of solvents and largely reduces the manual work, extracting and injecting the analytes in the chromatographic system automatically. Analysis, performed by GC/MS, ensures good and long lasting performance in separation and high specificity in the identification of the analytes. Two capillary chromatographic columns are proposed: the first to achieve a fast separation of o-C, the second, with a chiral stationary phase, to separate both o-C and m-C. The application of the assay to the determination of o-C and m-C in rotogravure printing workers and in subjects from the general population is reported.

Section snippets

Chemicals and standards preparation

o-C (99%+) (Sigma–Aldrich), m-C (99%) and p-C (99%) (Fluka) were used for standard preparation. o-Cresol-d8 (98 at.% D, Sigma–Aldrich) was used as internal standard. Hydrochloric acid aqueous solution (HCl, 37%, w/w), sodium hydroxide (NaOH), sodium chloride (NaCl), sodium acetate and acetic acid and methanol were purchased at a purification grade of 99% or higher from Carlo Erba Reagenti. Acetic acid and sodium acetate were used to prepare a 1 M buffer solution at pH 4.6 in water. Phenol-β-d

Chromatographic separation

In Fig. 1a and b the single ion mass-chromatograms of a standard solution containing 0.6 mg/l of o-C and m-C, obtained registering the ions m/z 108 (o-C and m-C, above) and 115 (o-cresol-d8, below) using a SupelcoWax10 column and a CP Cresol column, respectively, are reported. With the first column, the chromatographic peak of o-C was well resolved and quantified, while the peak of m-C was partially co-eluted with that of p-C and can be barely identified as a shoulder on the right side of it (

Discussion

The present work describes an original analytical procedure based on HS SPME GC/MS, useful to perform routine analysis of urinary cresols among subjects occupationally and non-occupationally exposed to toluene.

The major feature introduced by this assay, in comparison with the past procedures, is the use of SPME to extract the analytes from the headspace of specimen and directly inject them onto the chromatographic system. The advantages of using SPME are to skip tedious, time consuming and

References (35)

  • R.L. Dills et al.

    J. Chromatogr. B

    (1997)
  • M. Yoshikawa et al.

    J. Chromatogr.

    (1986)
  • J.B. Laurens et al.

    J. Chromatogr. B

    (2002)
  • K. Yamazaki et al.

    J. Forensic Sci. Soc.

    (1992)
  • G. Bieniek

    J. Chromatogr. B

    (1996)
  • K.B. Freeman et al.

    J. Chromatogr. B

    (1995)
  • S. Fustinoni et al.

    J. Chromatogr. B

    (1999)
  • IPCS, Environmental Health Criteria 52, Toluene, World Health Organization, Geneva,...
  • ACGIH, TLVs and BEIs based on the documentation of the threshold limit values for chemical substances and physical...
  • DFG, List of MAK and BAT values 2001, Report No. 37, WILEY-VCH, Weinhaim,...
  • Japan Society for Occupational Health (JSOH), J. Occup. Health 42 (2000)...
  • SCOEL, Recommendation from Scientific Committee on Occupational Exposure Limits for Toluene SCOEL/SUM/18H, European...
  • W. Woiwode et al.

    Br. J. Ind. Med.

    (1981)
  • K. Kono et al.

    Ind. Health

    (1985)
  • W. Woiwode et al.

    Arch. Toxicol.

    (1979)
  • M. Buratti et al.

    Med. Lav.

    (1989)
  • K. Nakashima et al.

    Analyst

    (1998)
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